Chemotaxis and phagocytosis are an integral part of immune response and play a key role in wound healing, angiogenesis, and embryogenesis. Found in all animals, including protozoa, these fundamental cellular processes seem to have remained closely associated and essentially unchanged throughout evolution. A singular G-protein beta-gamma-dimer is a central component in chemotactic sensing in D. discoideum; the powerful genetics available in these free-living cells provides a unique opportunity for analysis of these pathways. Our investigation of several related biochemical responses triggered by chemotactic and phagocytic stimuli is designed to elucidate the early steps in signal transduction. The pivotal role of the beta-gamma-dimer allows a molecular genetic approach to probe its structure and to examine its functions in vivo. Amino acid substitutions in the beta-subunit the conditionally block specific functions of the beta-gamma-dimer will be identified by random mutagenesis. The D. discoideum gamm- subunit(s) will be cloned and disrupted and mutant and wild-type beta-gamma-dimers will be overexpressed. Proteins that interact with beta-gamma-dimers will be cloned and disrupted and the biochemical events associated with chemotaxis and phagocytosis analysed. Chemoattractant mediated activation of adenylyl cyclase directly involves two novel cytosolic proteins, Crac and Pianissimo, in addition to heterotrimeric G-proteins. The interaction of these proteins with beta-gamma-dimers in vitro will be investigated. Genetic analysis of these genes in microorganisms-Pianissimo is an essential gene in S. cerevisiae-will be extended and the mammalian homologues will be identified. Cells lacking the beta-subunit are severly defective in phagocytosis as well as chemotaxis. The requirement for a G-protein linked co- stimulus in phagocytosis is facinating since these two processes share many features. An investigation of the block in phagocytosis is planned, focusing on the pathway linking external stimuli to actin polymerization. A novel screen of restriction enzyme mediated insertional (REMI) mutants is designed to isolate novel genes required for both chemotaxis and phagocytosis.